Difference between revisions of "Part:BBa K808032"
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Of course the first step for showing any kind of functionality is to perform a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page SDS PAGE]. ''E.coli'' Top10 is transformed with BBa-K808032 and induced at an OD<sub>600</sub>=0.5 with the arabinose concentrations of 0.02%, 0.2%, 0.5% and no arabinose at all (serving as a negative control). Incubation occured at 30°C over night. Afterwards the expressed chimeric protein gets [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification purified]. The resulting supernatant is loaded but the cell debris pellet is treated with an special detergent (n-Dodecyl β-D-maltoside) ,in order to solve our membrane bound protein, and is loaded as well. | Of course the first step for showing any kind of functionality is to perform a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page SDS PAGE]. ''E.coli'' Top10 is transformed with BBa-K808032 and induced at an OD<sub>600</sub>=0.5 with the arabinose concentrations of 0.02%, 0.2%, 0.5% and no arabinose at all (serving as a negative control). Incubation occured at 30°C over night. Afterwards the expressed chimeric protein gets [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification purified]. The resulting supernatant is loaded but the cell debris pellet is treated with an special detergent (n-Dodecyl β-D-maltoside) ,in order to solve our membrane bound protein, and is loaded as well. | ||
| − | [[File: | + | [[File: SDS Page of BBa_K808030.png]] |
* 2: 0.5% arabinose supernatant, 3: 0.5% arabinose treated pellet, 4: 0.2% arabinose supernatant, 5: 0.2% arabinose treated pellet, 7: 0.02% arabinose supernatant, 8: 0.02% arabinose treated pellet, 9: no arabinose supernatant, 10: no arabinose treated pellet | * 2: 0.5% arabinose supernatant, 3: 0.5% arabinose treated pellet, 4: 0.2% arabinose supernatant, 5: 0.2% arabinose treated pellet, 7: 0.02% arabinose supernatant, 8: 0.02% arabinose treated pellet, 9: no arabinose supernatant, 10: no arabinose treated pellet | ||
Revision as of 15:51, 23 September 2012
Status: 500 Content-type: text/html
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This is a composite of 2 different BioBricks formed by a standard assembly, forming a scar in between.
It contains the following units:
AraC-AraO2-AraO1-AraPromo(PBAD-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
which is similiar to BBa_K808000 & BBa_K808030
It is an operon, being induced by adding 0.02% to 0.5% of L-Arabinose. It is made for regulating the cell surface expression of our PET cleaving enzyme pNB-Est13, which is anchored C-terminal to EstA.
Usage and Biology
This part regulates the expression of our chimeric protein consisting of the following parts:
- PhoA signal sequence (BBa_K808028), which leads to periplasmatic expression.
- pNB-Est13 (BBa_K808026) is an enzyme that is able to hydrolyse PET
- EstA (BBa_K808027) is used as a C-terminal membrane anchor, being able to express even large enzymes on the bacterial surface
In order to get a more information about our composite part BBa_K808030 please take a look at its own registry page.
This site is about its expression rate and its activity in three different E.coli strains:
- [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10]
- [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5alpha]
- [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]
Functionality
The functionality of BBa_K808032 is shown by the following methods:
- SDS PAGE
- Western blot
- flow cytometry (after antibody staining)
- screening for hydrolysis by bacterial colonies using Tributyrin agar plates
- pNP-Assay with living cells using para-Nitrophenylbutyrate
SDS PAGE
Of course the first step for showing any kind of functionality is to perform a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page SDS PAGE]. E.coli Top10 is transformed with BBa-K808032 and induced at an OD600=0.5 with the arabinose concentrations of 0.02%, 0.2%, 0.5% and no arabinose at all (serving as a negative control). Incubation occured at 30°C over night. Afterwards the expressed chimeric protein gets [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification purified]. The resulting supernatant is loaded but the cell debris pellet is treated with an special detergent (n-Dodecyl β-D-maltoside) ,in order to solve our membrane bound protein, and is loaded as well.
- 2: 0.5% arabinose supernatant, 3: 0.5% arabinose treated pellet, 4: 0.2% arabinose supernatant, 5: 0.2% arabinose treated pellet, 7: 0.02% arabinose supernatant, 8: 0.02% arabinose treated pellet, 9: no arabinose supernatant, 10: no arabinose treated pellet
Western blot
In order to be sure of our protein being expressed we performed a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Western_Blot Western blot]. Our construct
Sequence and Features
Status: 500
Content-type: text/html
Software error:
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For help, please send mail to this site's webmaster, giving this error message and the time and date of the error.